Fig. 6: LIMK2-mediated downregulation of SPOP regulates c-Myc, AR and Arv7 levels and promotes tumorigenesis and EMT in vivo.

a LIMK2 augments c-Myc, ARv7 and AR protein levels in C4-2 cells. b WT-SPOP and 3A-SPOP decrease c-Myc levels. c Bar graph shows change in c-Myc protein level. The data depicted are mean ± SEM of three independent experiments. *P < 0.05 compared to C4-2 control cells. d 3A-SPOP promotes higher ubiquitination and degradation of c-Myc protein as compared to WT-SPOP. e Both WT-SPOP and 3A-SPOP decrease AR protein levels in C4-2 cells. f Histogram showing change in AR protein level. The data depicted are mean ± SEM of three independent experiments. *P < 0.05 compared to C4-2 control cells. g Ectopic expression of WT and 3A-SPOP promote robust ubiquitination and degradation of AR protein, as compared to control C4-2 cells. h Ectopic expression of 3A-SPOP decreases AR and ARv7 protein levels more significantly as compared to WT-SPOP in 22Rv1 cells. i Bar graph shows change in AR and ARv7 protein levels in 22Rv1, SPOP-22Rv1 and 3A-SPOP-22Rv1 cells. The data depicted are mean ± SEM of three experiments. *P < 0.05 compared to 22Rv1 control cells. j 3A-SPOP promotes more robust ubiquitination of AR in 22Rv1 cells as compared to WT-SPOP. k 3A-SPOP promotes more robust ubiquitination of ARv7 in 22Rv1 cells as compared to WT-SPOP. l AR binds SPOP in vector, SPOP and 3A-SPOP-expressing C4-2 cells. SPOP immune complexes were isolated from vector, SPOP and 3A-SPOP-C4-2 cells, followed by AR IB (left panel). Right panel depicts IgG IP in the same cell lines, followed by AR IB. Bottom panel depicts SPOP levels in the input. m WT and 3A-SPOP bind AR with equal efficiency. SPOP and AR association was examined using recombinant SPOP and AR in an in vitro pull-down assay. AR on beads was incubated with 6×-His-SPOP (WT and 3A) and binding was analysed. Right panel shows Coomassie staining of the input. n WT-SPOP inhibits tumour growth in vivo. Tumour growth curve of nude mice inoculated with C4-2 and SPOP-C4-2 cells on right and left shoulders, respectively. The mean value ± SEM were from three animals in each group. o Representative images of tumour-bearing nude mice. Pictures were taken 32 days following injection. p Tumour growth curve of C4-2 and 3A-SPOP-C4-2 cells on subcutaneous tumour growth in athymic nude mice. q Athymic nude mouse injected with C4-2 and 3A-SPOP-C4-2 cells on right and left shoulders, respectively. The images were taken 32 days after inoculation. A representative image is presented. r Expression of EMT markers in tumour tissues obtained from C4-2- and SPOP-C4-2 cell-injected mice. s Proposed model showing the consequences of LIMK2-SPOP signalling in CRPC pathogenesis.